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Alistair Chambers

Assistant Professor, Faculty of Medicine & Health Sciences

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Biography

BSc University of Durham 1983; PhD University of Warwick 1987, Postdoctoral Researcher, University of Oxford 1986-90, Lecturer, University of Nottingham 1990.

Research Summary

Telomeres and telomere proteins in fission yeast: Telomeres are the very ends of the linear DNA molecules that comprise eukaryotic chromosomes. In many organisms they consist of multiple repeats of a… read more

Recent Publications

Current Research

Telomeres and telomere proteins in fission yeast: Telomeres are the very ends of the linear DNA molecules that comprise eukaryotic chromosomes. In many organisms they consist of multiple repeats of a short DNA sequence rich in T and G bases in the DNA strand running 5' to 3' towards the chromosome end. This strand overlaps its partner strand at the end of the chromosome and forms a single stranded DNA extension. Both the double stranded region containing the repeats and the single stranded overhangs are the targets for sequence-specific DNA binding proteins. These proteins are of great interest because they play key roles in regulating the length and chromatin structure of the telomeres, with implications for cell ageing and cancer biology.

Telomeres and telomere proteins in fission yeast: Telomeres are the very ends of the linear DNA molecules that comprise eukaryotic chromosomes. In many organisms they consist of multiple repeats of a short DNA sequence rich in T and G bases in the DNA strand running 5' to 3' towards the chromosome end. This strand overlaps its partner strand at the end of the chromosome and forms a single stranded DNA extension. Both the double stranded region containing the repeats and the single stranded overhangs are the targets for sequence-specific DNA binding proteins. These proteins are of great interest because they play key roles in regulating the length and chromatin structure of the telomeres, with implications for cell ageing and cancer biology. Fission yeast (S. pombe) is a single cell eukaryote very distantly related to budding yeast (S. cerevisiae). It has three chromosomes which end in about 300 base pairs of repeat DNA containing multiple copies of the degenerate sequence TTACA(0-1)G(1-6). I am interested in the proteins that interact with this DNA and the mechanisms by which they mediate the properties of the telomeres. Initially we identified two sequence specific telomere binding proteins in fission yeast, as shown below (Duffy and Chambers, 1996):

Figure 1. A band shift assay using a labelled fragment of telomere DNA and a fission yeast protein extract. The complexes (bands I, IIa and IIb) are formed by two different DNA binding proteins.

One of these proteins is Taz1 (responsible for complexes IIa and IIb above), a key protein that negatively regulates telomere length and promotes the formation of a chromatin structure repressive for gene transcription and characteristic of chromosome ends (Cooper et al., 1997). Taz1 binds directly to DNA as a dimer (Spink et al., 2000). Two forms of Taz1 of different molecular weight can be identified in protein extracts, which probably accounts for the formation of the two complexes shown above (Spink et al., 2005). We are currently trying to understand the nature and significance of these two forms.

In a two-hybrid screen searching for proteins that interact with Taz1, we identified Pmt3, the fission yeast homologue of SUMO-1, as a binding partner. In collaboration with Jenny Ho and Felicity Watts at the University of Sussex we went on to show that Taz1 can be modified by Pmt3, although it was not possible to isolate a population of Taz1 molecules with this modification directly from fission yeast cells (Spink et al., 2005). Taz1 contains one good candidate for a Pmt3 modification site, located within the DNA binding region of the protein. We are currently mapping the site of Pmt3 modification within Taz1 and trying to understand the role this modification plays in regulating telomere function.

Figure 2. pmt3+ and pmt3- cells containing GFP-Taz1 were cultured and examined using DIC and fluorescence microscopy. Panels A and B show pmt3+ cells, C and D show pmt3- cells.

One approach is to use a GFP-Taz1 fusion protein to study the localisation of Taz1 in pmt3+ and pmt3- cells at different stages of the cell cycle. An example of this is shown above where the telomeres are visible as bright dots because of the associated GFP-Taz1. Note how abnormal the pmt3- cells (C) look compare to the pmt3+ cells (A).

The second telomere protein (responsible for complex I in Figure 1.) was purified using heparin agarose and telomere affinity chromatography. It was shown to have a molecular weight of about 50 kDa which is consistent with that of S. pombe Tbf1, the fission yeast homologue of an essential protein present in budding yeast which resembles mammalian telomere binding factors (Pitt et al., 2007). We are studying this protein to try to understand why it is essential, whether it has roles at other sites in the genome, which proteins it interacts with and what functions it performs at telomeres.

Cooper, J.P., Nimmo, E.R., Allshire, R.C. and Cech, T.R. 1997. Regulation of telomere length and function by a myb-domain protein in fission yeast. Nature 385, 744-747.

Duffy, M. and Chambers, A. 1996. DNA-protein interactions at the telomeric repeats of Schizosaccharomyces pombe, Nucleic Acids Research 24, 1412-1419.

Pitt, C.W., Valente, L.P., Rhodes, D. and Simonsson, T. 2008. Identification and characterization of an essential telomeric repeat binding factor in fission yeast. Journal of Biological Chemistry 283, 2693-2701.

Spink, K.G., Evans, R.J. and Chambers, A. 2000. Sequence-specific binding of Taz1p dimmers to fission yeast telomeric DNA. Nucleic Acids Research 28, 527-533.

Spink, K., Ho, J.C.Y., Tanaka, K., Watts, F.Z. and Chambers, A. 2005. The telomere-binding protein taz1p as a target for modification by a SUMO-1 homologue in fission yeast. Biochemical Genetics 43, 103-117.

Future Research

Fission yeast (S. pombe) is thought to have diverged from budding yeast (S. cerevisiae) between 330 and 420 million years ago. It has been used widely in studies of the eukaryotic cell cycle and as a model for aspects of chromosome biology. Fission yeast has three chromosomes with large and complex centromeres similar to those in higher eukaryotes. We are studying the proteins that interact with the ends of these chromosomes, the telomeres, in order to compare them with those in budding yeast and mammalian cells. Key aspects of the protein complexes assembled at fission yeast telomeres resemble those established at the ends of human chromosomes. The major telomere DNA binding protein in fission yeast is Taz1p (Cooper et al. 1997). Taz1p binds to DNA as a preformed homodimer (Spink et al. 2000) that then targets a second factor, Rap1p, to the telomere via a protein:protein interaction (Chikashige and Hiraoka 2001; Kanoh and Ishikawa 2001). In mammalian cells, a factor called TRF2 targets Rap1p in a similar way (Li et al. 2000). We are using genetic and biochemical approaches to characterise the interactions between fission yeast telomere proteins in order to understand how the protein complexes found at telomeres are assembled and to gain insights into their conserved functional roles.

School of Life Sciences

University of Nottingham
Medical School
Queen's Medical Centre
Nottingham NG7 2UH

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